The present invention relates to the calibration, standardization, use and testing of microscopes. More particularly, the present invention relates to a test slide and to methods of production and use thereof.
As is known, microscopes must be tested to ensure that the images observed with the microscope are accurate and/or understood. In particular, the resolving power, the image formation capabilities and the aberrations of microscopes and their related optics must be determined and documented before reliance can be placed on the observed images obtained therewith. Generally, the testing of a microscope can comprise a set of one or more characterizations and/or calibrations. For example, characterization tests can be performed to determine the depth of field, flatness of field, aberrations and illumination characteristics within the field of view of a microscope at various magnification settings. Also, calibration tests can be performed to determine the actual magnification levels, measure the amount of any observed aberrations, etc.
Conventionally, microscopists and microscope manufacturers have experienced difficulty in locating naturally occurring or artificial structures with suitable known and reproducible features to employ as test specimens for microscope testing. It has also been difficult to find test specimens that yield information on the means by which microscopes form their images. This latter factor is important as these means often have limitations such that the resolution and imaging of objects with differing geometries may not be comparable, due to the methods of image formation or physical limits of the optics in the microscope. In some cases it has been noted that seemingly properly formed images will in fact be completely erroneous due to effects of optical interference or limits in the optics due to aberrations, etc.
Early previous attempts to manufacture suitable test systems have primarily consisted of ruled gratings of uniformly spaced parallel lines scribed in the surface of a substrate by a stylus, such as a diamond point. These ruled substrates have been produced to address the need for resolution testing standards for light microscopes and such gratings are commonly employed as diffraction gratings for spectroscopic applications.
Such gratings were developed most notably by F. A. Norbert in Germany in the mid to late 1800""s, as described in xe2x80x9cThe Great Age of the Microscopexe2x80x9d, Gerard L""E Turner, 1989, First Edition, as published by the Royal Microscopical Society, pages 344-345 and in xe2x80x9cMicrographia Historicaxe2x80x94The Study of the History of the Microscopexe2x80x9d, by the same author, 1972, published by the Royal Microscopical Society, pages 20-24.
Norbert manufactured ruled gratings that were used in tests of microscope objective performance and he eventually ruled a grating with a spacing between the grating lines of 0.13 microns (i.e.xe2x80x94from the trough of one line to the trough of the next line), although the proof of this spacing had to await the development of the electron microscope as this spacing was too small to be resolved using traditional light microscope means.
Norbert""s standard offering was produced as a series of graded gratings ranging from a course grating to a fine grating arranged on a cover slip which was then mounted to a standard microscope slide. These slides were sold as test slides and became a relatively accepted means of comparing the performance of microscopes.
Since Norbert""s time, there have been few other attempts to produce such similar test slides and there has been no attempt of which the present inventor is aware to produce a comprehensive industry-standard test slide. It should also be noted that diffraction gratings are still not commercially available with sufficiently close spacings to test the resolving power of the best light microscopes which are now available.
Instead, the standard means of testing microscopes at the time of Norbert, and one still in use today, is to use a diatom as a test specimen, as diatoms have periodic structures as part of their features and these periodic structures provide a somewhat known set of optical characteristics and geometry. The two most common diatoms used for this purpose are the Amplipleura Pellucida and Pleurosigma Angulatum. While commonly employed as test specimens, diatoms still leave much to be desired as there is some variation from diatom to diatom and their exact appearance is not known, rendering their use for calibration and characterization purposes difficult at best.
Most recently, nanofabrication techniques have been employed to produce test slides and these are described by Rudolf Oldenburg et al. in, xe2x80x9cStandard Test Targets For High Resolution Light Microscopyxe2x80x9d, Nanofabrication and Biosystems, Cambridge University Press, 1996, pages 123-138, and in xe2x80x9cImage sharpness and contrast transfer in coherent confocal microscopyxe2x80x9d, Journal of the Royal Microscopical Society, 172, pages 31-39, 1993. These references describe slides with test images produced by the use of focused electron beam directwrite photo-lithography techniques and direct etch micromachining techniques, the slides being useful for visible light microscopy.
However, several problems exist with the Oldenburg slides. For example, the slides are only useful with visible light microscopes and are not suitable for UV, IR, etc. microscopes. Also, due to the method of their fabrication, these slides are very expensive and time consuming to manufacture. Also, the test patterns employed on these slides are limited to artificial constructs such as Siemens stars, gratings, etc.
It is desired to have a test system which is superior to the prior art systems described above and which produces an accurate and repeatable means of comparing microscope performance and of assessing image formation capabilities and image formation theory.
It is an object of the present invention to provide a novel test slide for microscopes. It is a further object of the present invention to provide a novel method of manufacturing a test slide for microscopes.
According to a first aspect of the present invention, there is provided a test slide comprising:
a substrate;
a test pattern formed on said substrate, said pattern comprising at least a diffraction grating and a scale system; and
a locating pattern formed on said substrate to assist an observer to locate said test pattern.
According to another aspect of the present invention, there is provided a method of manufacturing a test slide, comprising the steps of:
(i) providing a substrate with an image area;
(ii) coating at least a portion of said image area with a resist compound;
(iii) exposing said resist compound to form a test pattern and a locating pattern thereon;
(iv) developing said resist compound and removing portions of said resist compound from said substrate to form said test pattern and a locating pattern thereon.
According to yet another aspect of the present invention, there is provided a test slide comprising:
a substrate including an image area;
a test pattern formed on said substrate in said image area, said pattern comprising a known image having at least two or more features selected from the group comprising grating-type structures, scale systems, image series, offset segment pie stars and indicia to uniquely identify said slide.
According to yet another aspect of the present invention, there is provided a test slide comprising:
a substrate;
a test pattern formed on said substrate, said pattern having known shape and size; and
a protective layer on said test pattern, said layer inhibiting inadvertent damage to said test pattern and being non-opaque to an preselected range of wavelengths for which said slide is intended to be used.
According to yet another aspect of the present invention, there is provided a method of forming a test slide for microscopes, comprising the steps of:
(i) forming a master test pattern on an information carrier for an injection molding device, said test pattern including patterns of known size and shape;
(ii) inserting said information carrier into said injection molding device;
(iii) cycling said injection molding device to inject liquefied resin into contact with said information carrier and to cool said resin to form a plastic carrier with said test pattern formed in one surface; and
(iv) removing said plastic carrier from said injection molding machine.
According to yet another aspect of the present invention, there is provided a method of forming a test slide for microscopes, comprising the steps of:
(i) forming a master test pattern on an information carrier for a mold, said test pattern including patterns of known size and shape;
(ii) inserting said information carrier into a mold;
(iii) adding a substrate material to said mold to contact said information carrier and setting said substrate material to form a carrier with said test pattern formed in one surface; and
(iv) removing said carrier from said mold.